Funduscopy in adult zebrafish and its application to isolate mutant strains with ocular defects

Funduscopy is one of the most commonly used diagnostic tools in the ophthalmic practice, allowing for a ready assessment of pathological changes in the retinal vasculature and the outer retina. This non-invasive technique has so far been rarely used in animal model for ophthalmic diseases, albeit its potential as a screening assay in genetic screens. The zebrafish (Danio rerio) is well suited for such genetic screens for ocular alterations. Therefore we developed funduscopy in adult zebrafish and employed it as a screening tool to find alterations in the anterior segment and the fundus of the eye of genetically modified adult animals.A stereomicroscope with coaxial reflected light illumination was used to obtain fundus color images of the zebrafish. In order to find lens and retinal alterations, a pilot screen of 299 families of the F3 generation of ENU-treated adult zebrafish was carried out.Images of the fundus of the eye and the anterior segment can be rapidly obtained and be used to identify alterations in genetically modified animals. A number of putative mutants with cataracts, defects in the cornea, eye pigmentation, ocular vessels and retina were identified. This easily implemented method can also be used to obtain fundus images from rodent retinas.In summary, we present funduscopy as a valuable tool to analyse ocular abnormalities in adult zebrafish and other small animal models. A proof of principle screen identified a number of putative mutants, making funduscopy based screens in zebrafish feasible.

Four-year results following treatment of intrabony periodontal defects with an enamel matrix derivative alone or combined with a biphasic calcium phosphate

The aim of this study was to evaluate the 4-year clinical outcomes following regenerative surgery in intrabony defects with either EMD + BCP or EMD. Twenty-four patients with advanced chronic periodontitis, displaying one-, two-, or three-walled intrabony defect with a probing depth of at least 6 mm, were randomly treated with either EMD + BCP (test) or EMD alone (control). The following clinical parameters were evaluated at baseline, at 1 year and at 4 years after regenerative surgery: plaque index, gingival index, bleeding on probing, probing depth, gingival recession, and clinical attachment level (CAL). The primary outcome variable was CAL. No differences in any of the investigated parameters were observed at baseline between the two groups. The test group demonstrated a mean CAL change from from 10.8 ± 1.6 mm to 7.4 ± 1.6 mm (p < 0.001) and to 7.6 ± 1.7 mm (p < 0.001) at 1 and 4 years, respectively. In the control group, mean CAL changed from 10.4 ± 1.3 at baseline to 6.9 ± 1.0 mm (p < 0.001) at 1 year and 7.2 ± 1.2 mm (p < 0.001) at 4 years. At 4 years, two defects in the test group and three defects in the control group have lost 1 mm of the CAL gained at 1 year. Compared to baseline, at 4 years, a CAL gain of ≥3 mm was measured in 67% of the defects (i.e., in 8 out of 12) in the test group and in 75% of the defects (i.e., in 9 out of 12) in the control group. There were no statistically significant differences in any of the investigated parameters at 1 and at 4 years between the two groups. Within their limits, the present results indicate that: (a) the clinical improvements obtained with both treatments can be maintained over a period of 4 years, and (b) in two- and three-walled intrabony defects, the addition of BCP did not additionally improve the outcomes obtained with EMD alone. In two- and three-walled intrabony defects, the combination of EMD + BCP did not show any advantage over the use of EMD alone.

Wound healing following regenerative procedures in furcation degree III defects: histomorphometric outcomes

Degree III furcation involvements were surgically created at four first molars in each of three monkeys. Following 6 weeks of healing, full-thickness flaps were elevated. Following 24% EDTA gel conditioning, the defects were treated with one of the following: (1) enamel matrix proteins (EMD), (2) guided tissue regeneration (GTR) or (3) a combination EMD and GTR. The control defects did not receive any treatment. After 5 months of healing, the animals were sacrificed. Three 8 μm thick histological central sections, 100 μm apart, were used for histomorphometric analysis in six zones of each tooth either within the furcation area or on the pristine external surface of the root. In all specimens, new cementum with inserting collagen fibres was formed. Following GTR or GTR + EMD, cementum was formed up to and including the furcation fornix indicating complete regeneration on the defect periphery. Periodontal ligament fibres were less in all four modalities compared to pristine tissues. In the teeth treated with GTR and GTR + EMD a higher volume of bone and periodontal ligament tissues was observed compared to EMD. After 5 months of healing, regenerated tissues presented quantitative differences from the pristine tissues. In the two modalities where GTR alone or combined with EMD was used, the regenerated tissues differed in quantity from the EMD-treated sites.

Reconstruction of large supra-eyebrow and forehead defects using the hatchet flap principle and sparing sensory nerve branches

To reconstruct a forehead defect, a plastic surgeon must be knowledgeable about the neural, vascular, and muscular anatomy. The position of fixed structures such as eyebrows and hairline should be respected. For the past 5 years, we have used double hatchet flaps for reconstruction of relatively large supra-eyebrow and forehead defects. Because this flap does not appear to be among the techniques used by young plastic surgeons, we thought that it would be valuable to report our experience.

Repair of superficial osteochondral defects with an autologous scaffold-free cartilage construct in a caprine model: implantation method and short-term results

OBJECTIVE: To compare four different implantation modalities for the repair of superficial osteochondral defects in a caprine model using autologous, scaffold-free, engineered cartilage constructs, and to describe the short-term outcome of successfully implanted constructs. METHODS: Scaffold-free, autologous cartilage constructs were implanted within superficial osteochondral defects created in the stifle joints of nine adult goats. The implants were distributed between four 6-mm-diameter superficial osteochondral defects created in the trochlea femoris and secured in the defect using a covering periosteal flap (PF) alone or in combination with adhesives (platelet-rich plasma (PRP) or fibrin), or using PRP alone. Eight weeks after implantation surgery, the animals were killed. The defect sites were excised and subjected to macroscopic and histopathologic analyses. RESULTS: At 8 weeks, implants that had been held in place exclusively with a PF were well integrated both laterally and basally. The repair tissue manifested an architecture similar to that of hyaline articular cartilage. However, most of the implants that had been glued in place in the absence of a PF were lost during the initial 4-week phase of restricted joint movement. The use of human fibrin glue (FG) led to massive cell infiltration of the subchondral bone. CONCLUSIONS: The implantation of autologous, scaffold-free, engineered cartilage constructs might best be performed beneath a PF without the use of tissue adhesives. Successfully implanted constructs showed hyaline-like characteristics in adult goats within 2 months. Long-term animal studies and pilot clinical trials are now needed to evaluate the efficacy of this treatment strategy.

An in vivo study of a growth-factor enhanced, cell free, two-layered collagen-tricalcium phosphate in deep osteochondral defects

Focal osteochondral defects are still a challenging problem in joint surgery. We have developed a two-layered implant consisting of a basal porous beta-tricalcium phosphate (TCP) for bone reconstruction and a superficial fibrous collagen type I/III layer for cartilage regeneration. Fifty-four osteochondral defects in the trochlear groove of 27 Göttinger Minipigs were created and either left untreated, treated with the implant alone, or the implant augmented with an additional growth factor mixture, which was assumed to stimulate cell and tissue differentiation. Follow-up was 6, 12 and 52 weeks with n=6 for each group. The repair tissue was evaluated for its gross appearance and biomechanical properties. Histological sections were semi-quantitatively scored for their histomorphological structure. Treatment with the two-layered implant improved defect filling and subchondral bone repair at 6 and 12 weeks follow-up. The TCP was replaced by cancellous bone at 52 weeks. Cartilage repair tissue mainly consisted of fibrocartilage and showed a moderate cell density up to the joint surface. Growth factor treatment improved the mechanical and histomorphological properties of the cartilage repair tissue at 12, but not at 52 weeks postoperatively. In conclusion, the two-layered collagen-TCP implant augmented with chondroinductive growth factors seems a promising new option for the treatment of deep osteochondral defects in joint surgery.

Effect of Bio-Oss on osseointegration of dental implants surrounded by circumferential bone defects of different dimensions: an experimental study in the dog

OBJECTIVES: This study was designed to evaluate the effect of gap width and graft placement on bone healing around implants placed in simulated extraction sockets of various widths in four Labrador dogs. MATERIALS AND METHODS: Five Osseotite implants per dog were placed in the mandible of four dogs. Two implants were inserted into sites with a 2.37 mm and two with a 1 mm gap present between the implants and bone around the coronal 6 mm of the implants in each dog. For one of each gap sizes, the gap was filled with Bio-Oss, and the other two with blood alone. A fifth implant was inserted without a gap and used as a control. Ground sections were prepared from biopsies taken at 4 months and histometric measurements of osseointegration and bone between the threads made for the coronal 6 mm. RESULTS: The medians for osseointegration ranged from 5.2 mm for control to 1-2.6 mm for the test modalities. There were significant differences for linear measurements of osseointegration (chi(2) 18.27; df 4; P=0.0011) and bone area within threads (chi(2) 23.4; df 4; P=0.0001) between test modalities. CONCLUSIONS: The results suggest that the wider the gap around the implants, the less favourable the histological outcome at short time intervals following treatment. They also infer that bone grafting with an organic bovine bone xenograft seems to lead to a more favourable histological outcome for wider circumferential defects but not for narrower defects.

Microbial colonization patterns predict the outcomes of surgical treatment of intrabony defects

AIM: To explore the impact of bacterial load and microbial colonization patterns on the clinical outcomes of periodontal surgery at deep intrabony defects. MATERIALS AND METHODS: One hundred and twenty-two patients with advanced chronic periodontitis and at least one intrabony defect of >3 mm were recruited in 10 centres. Before recruitment, the infection control phase of periodontal therapy was completed. After surgical access and debridement, the regenerative material was applied in the test subjects, and omitted in the controls. At baseline and 1 year following the interventions, clinical attachment levels (CAL), pocket probing depths (PPD), recession (REC), full-mouth plaque scores and full-mouth bleeding scores were assessed. Microbial colonization of the defect-associated pocket was assessed using a DNA-DNA checkerboard analysis. RESULTS: Total bacterial load and counts of red complex bacteria were negatively associated with CAL gains 1 year following treatment. The probability of achieving above median CAL gains (>3 mm) was significantly decreased by higher total bacterial counts, higher red complex and T. forsythensis counts immediately before surgery. CONCLUSIONS: Presence of high bacterial load and specific periodontal pathogen complexes in deep periodontal pockets associated with intrabony defects had a significant negative impact on the 1 year outcome of surgical/regenerative treatment.

Guided tissue regeneration/deproteinized bovine bone mineral or papilla preservation flaps alone for treatment of intrabony defects. II: radiographic predictors and outcomes

OBJECTIVES: This study reports the secondary analysis of a randomized-controlled clinical trial designed to assess the efficacy of deproteinized bovine mineral and a collagen membrane in the treatment of intrabony defects. The specific aims of this report are (1) to analyse the radiographic bone changes 1 year after therapy and (2) to assess the association between radiographic defect angle and treatment outcomes. MATERIALS AND METHODS: Baseline and 12-month radiographs were collected from 120 patients with advanced chronic periodontitis from 10 centres in seven countries as part of a multi-centre clinical trial. All patients had at least one intrabony defect > or =3 mm in depth. The treatment consisted of simplified or modified papilla preservation flaps to access the defect. After debridement of the area, a deproteinized bovine mineral and a collagen membrane were applied in the test subjects, and omitted in the controls. Main outcome measures were radiographic bone fill and defect resolution 1 year after surgery. RESULTS: One hundred and twenty pairs of radiographs were obtained, of which 110 pairs were measurable (57 tests and 53 controls). One year after treatment, radiographic resolution of the intrabony component was significantly higher in the test group (3.2+/-1.7 mm) when compared with the controls (1.7+/-1.9 mm). Multivariate analysis indicated that the treatment and the baseline radiographic depth of the intrabony defect significantly influenced the radiographic bone fill of the intrabony defect 1 year following treatment. The percentage of resolution of the defect was influenced by the treatment provided and the baseline plaque score. The baseline radiographic defect angle did not show a significant impact on the clinical and radiographic outcomes. CONCLUSIONS: Regenerative periodontal surgery with a deproteinized bovine bone mineral and a collagen membrane offered additional benefits in terms of radiographic resolution of the intrabony defect and predictability of outcomes with respect to papilla preservation flaps alone.

Enhancing sealing of fetal membrane defects using tissue engineered native amniotic scaffolds in the rabbit model

OBJECTIVE: The purpose of this study was to compare the efficacy of native engineered amniotic scaffolds (AS) and polyesterurethane scaffolds (DegraPol) and document wound healing response when sealing iatrogenic fetal membrane defects in the rabbit model. STUDY DESIGN: Native AS were engineered from freshly harvested membranes of 23 days' gestational age (GA; term = 31-2 d). Acellularity of AS was assessed by histology, light and scanning electron microscopy. Fetal membrane defects were created by 14 gauge-needle puncture at GA 23 days and primarily closed with AS (n = 10) or DegraPol (n = 10) or left unclosed (positive controls; n = 10). Sixty-one sacs served as negative controls. At GA 30 days a second look hysterotomy was performed to assess presence of amniotic fluid (AF) and harvest plugging sites for microscopic evaluation. RESULTS: Engineered AS had a cell-free collagenous fiber network. AF was significantly higher only in the DegraPol group (78%; P < .05) compared to the AF in positive controls (17%). Integration of plugs in the fetal membrane defect was better with AS than DegraPol, with higher reepithelialization rates (AS: 52.5% +/- 6.5%; DegraPol: 11.6% +/- 2.6%; P < .001) and proliferation indices (AS: 0.47 +/- 0.03; DegraPol: 0.28 +/- 0.04; P = .001). In both treatment groups, cell proliferation in the myometrium was increased (P < .05). CONCLUSION: Native AS seal iatrogenic fetal membrane defects better than DegraPol. Within a week, there is abundant reepithelilization and minimal local inflammation. This yields the proof of principle that engineered native, amniotic membrane scaffolds enhance fetal membrane wound healing response.

Enhanced early chondrogenesis in articular defects following arthroscopic mesenchymal stem cell implantation in an equine model

Mesenchymal stem cells (MSCs) provide an important source of pluripotent cells for musculoskeletal tissue repair. This study examined the impact of MSC implantation on cartilage healing characteristics in a large animal model. Twelve full-thickness 15-mm cartilage lesions in the femoropatellar articulations of six young mature horses were repaired by injection of a self-polymerizing autogenous fibrin vehicle containing mesenchymal stem cells, or autogenous fibrin alone in control joints. Arthroscopic second look and defect biopsy was obtained at 30 days, and all animals were euthanized 8 months after repair. Cartilage repair tissue and surrounding cartilage were assessed by histology, histochemistry, collagen type I and type II immunohistochemistry, collagen type II in situ hybridization, and matrix biochemical assays. Arthroscopic scores for MSC-implanted defects were significantly improved at the 30-day arthroscopic assessment. Biopsy showed MSC-implanted defects contained increased fibrous tissue with several defects containing predominantly type II collagen. Long-term assessment revealed repair tissue filled grafted and control lesions at 8 months, with no significant difference between stem cell-treated and control defects. Collagen type II and proteoglycan content in MSC-implanted and control defects were similar. Mesenchymal stem cell grafts improved the early healing response, but did not significantly enhance the long-term histologic appearance or biochemical composition of full-thickness cartilage lesions.